Cholesterol, Bile, and Blood Lipoproteins

Question 1

Describe the key features of the structure of cholesterol and its ester.

Answer 1

Cholesterol:

• Steroid nucleus: 4 planar hydrocarbon rings
• 8 carbon hydrocarbon tail attached to carbon 17 of D ring
• Hydroxyl group attached to carbon 3 of ring A
• Double bond between carbons 5-6 of B ring

Cholesterol Ester:

-Esterified to fatty acid at carbon 3

Question 2

What is the function of cholesterol in cell membranes?

Answer 2

The steroid nucleus intercalates between fatty acid chains of phospholips. This increases mechanical strength, decreases membrane fluididty.

Question 3

What are the relative amounts of dietary cholesterol and phytosterol absorbed by humans?

Answer 3

• 40% of dietary cholesterol
• 5% phytosterols

Question 4

What is the cause of Sitosterolemia? Outcomes?

Answer 4

• Autosomal recessive plant sterol storage disease
• Mutation in ABCG5 and ABCG8 genes for sterol transporters sterolin 1 and 2.
• Less phytosterols pumped into the intestinal lumen, less excretion from liver.
• Phytosterols accumulate causing xanthomas, premature atheroschlerosis.

Question 5

Where in the body are the major sites of cholesterol synthesis?

Answer 5

• Liver, intestines, adrenal cortex, reproductive tissues.

Question 6

Where in the cell are the reactions of cholesterol synthesis carried out?

Answer 6

The cytoplasmic side of the smooth ER membrane.

Question 7

Describe the first two steps of cholesterol synthesis.

Answer 7

Question 8

Describe the rate limiting step of cholesterol synthesis.

Answer 8

Question 9

Which hormones regulate HMG CoA reductase and how?

Answer 9

Hormones regulate the expression of its gene:

• Insulin and thyroxine upregulate expression.
• glucagon and glucocorticoids down-regulate expression.

Question 10

Describe the enzymatic degredation of HMG-CoA reductase.

Answer 10

High cholesterol levels result in binding of cholesterol to the sterol-sensing domain of the reductase itself. This causes binding of the reductase to insigs in the ER membrane which triggers ubiquitination and proteasomal degradation of the enzyme.

Question 11

How is AMP related to the regulation of HMG-CoA reductase?

Answer 11

AMP activates a protien kinase which phosphorylates the reductase deactivating it. High AMP -> low activity.

Question 12

How do statin drugs work?

Answer 12

Question 13

What is the mechanism of HMG-CoA Reductase gene expression regulation? How is the level of cholesterol related?

Answer 13

Expression of the HMG CoA Reductase gene is under the control of a transcription factor SREBP-2 (sterol regulatory element binding protein-2) which binds the cis acting sterol regulatory element (SRE). SREBP-2, in its inactive form, is an integral ER membrane protein. It associates with another ER protein SCAP (SREBP cleavage activating protein). When cholesterol levels are low the SREBP-2-SCAP complex moves to the Golgi where it stimulates the specific cleavage of SREBP resulting in a soluble fragment that is the activated SREBP transription factor. The SREBP transcription factor enters the nucleus, binds SRE and stimulates the expression of HMG CoA Reductase mRNA transcripts, increasing expression of the enzyme and cholesterol synthesis. When cholesterol is at high concentration it binds to the sterol sensing domain of SCAP, which binds to additional ER proteins (insigs, insulin induced gene
products) which anchor the SREBP-2-SCAP complex to the ER membrane. As a result cholesterol synthesis decreases.

Question 14

Name and describe the structure of the two major primary bile acids/salts.

Answer 14

• The two major primary bile acids are shown below. Bile salts are the deprotonated form.
• Bile salts and acids have their OH- groups below the plain of the sterol ring structure and their methyl groups above. The result is that the bile acids and salts have a polar face and a nonpolar face.

Question 15

-Describe the steps of bile acid synthesis, especially the RLS.

Answer 15

• OH groups are added to the sterol ring structure, the double bond in the B ring is reduced, and the hydrocarbon chain is shortened introducing a carboxyl group to the end of the chain.
• Hydroxylation of carbon 7 of cholesterol is the rate limiting step. bile acids down regulate the expression of cholesterol 7-alpha-hydroxylase. (see image.)

Question 16

What are the conjugated bile salts? What is the purpose of conjugation of bile salts?

Answer 16

• Before bile acids leave the liver they are conjugated to either to glycine or taurine forming: glycocholic, glycochenocholic, taurocholic, and taurochenocholic acids. (see image.) The ratio of glycine to taurine forms is 3:1.

-The addition of carboxyl group (glycine) or a sulfate group (taurine)
lower the pKa and are therefore the bile salts are fully ionized at the
alkaline pH of bile. This makes them better detergents (they are more amphipathic)

Question 17

What effects can the intestinal flora have on secreted conjugated bile salts?

Answer 17

• They can remove glycine or taurine producing primary bile salts/acids.
• They can remove the carbon 7 hydroxyl to produce secondary bile salts/acids.

Question 18

Describe the process of enterohepatic circulation.

Answer 18

• Bile salts from the liver hepatocytes are released into the bile.
• They pass through the bile duct to the duodenum (some are decarboxylated/deconjugated.)
• They move to the terminal ileum where ~95% are reabsorbed daily. Na+-bile salt cotransporters mediate the reabsorption.
• Albumin acts as a carrier for the bile salts/acids in the blood while they return to the liver hepatocytes via the portal vein.

Question 19

What is the aproximate amount of bile salts excreted in feces each day? How is this loss compesensated for?

Answer 19

-The 0.5 grams/day of primary and secondary bile salts (<3%) lost in the feces is compensated for by the 0.5 grams/day synthesized from cholesterol in the liver.

Question 20

What is the basic etiology of gall stone formation? What is the medical term for gall stone formation?

Answer 20

• A disruption causing a decrease of bile salt secretion or increased cholesterol secretion can causes an imbalance in the bile where cholesterol cannot be sufficiently solubilized by the bile salts and phospholipids.
• The result is the precipitation of cholesterol and formation of gall stones (“cholelithiasis”.)

Question 21

Describe the structure and function of lipoprotein lipase (LPL.)

Answer 21

• LPL is an anti-parallel homodimer attached by heparin sulfate to the capillary walls in most tissues.
• Each subunit contains an Nterminal domain that contains the lypolytic site and a Cterminal domain that binds to the lipoprotein particle and gives substrate specificity
• Upon binding of ApoC-II, the C-terminal region supplies the lipid in the lipoprotein to a lid covering a hydrophobic active site in the N-terminal domain. The lid moves so that the TAG can be degraded.

Question 22

Describe the basic structure of a lipoprotein.

Answer 22

• Lipoproteins contain an inner hydrophobic core composed of triacyl- glycerol (TAG) and cholesterol esters.
• This hydrophobic lipid core is surrounded by a shell containing: amphipathic phospholipids with their polar head groups facing the aqueous exterior, unesterified cholesterol, and apoliproteins.

Question 23

What lab techniques are available for the separation of blood lipoproteins? Describe the stratification observed in each form of separation.

Answer 23

-Lipoprotein particles can be separated based on electrophoretic
mobility (combination of size and charge) or based on their density by ultracentrifugation.

-see image for stratification.

Question 24

How are insulin levels related to LPL?

Answer 24

In the fed state (elevated insulin) adipose tissue LPL expression is increased and muscle LPL is decreased. The opposite occurs in the fasted state.

Question 25

type 1 hyperlipoproteinemia (subtypes, common complications)

Answer 25

Subtypes:

• 1a: deficiency in LPL
• 1b: deficiency in ApoC-II

Complications:

• High circulating TAG, chylomicrons
• Recurrent pancreatitis

Question 26

Summarize the synthesis of a chylomicron.

Answer 26

Apo B-48 is synthesized and glycosylated in intestinal mucosal cells. Assembly of the nascent chylomicron requires microsomal triglyceride transfer protein (MTP) which loads Apo B-48 with lipid. The particle is then transferred from the ER to Golgi and is packaged in a secretory vesicles. The secretory vesicle then fuse with the plasma membrane releasing the nascent chylomicron which enters the lymphatic system and then the blood.

Question 27

Describe the life cycle of a newly formed chylomicron.

Answer 27

• When a nascent chylomicron reaches the blood it receives Apo-E and Apo-Cs including apo C-II from HDL particles.
• Lipoprotein lipase (LPL) is activated by apo C-II. LPL hydrolyzes the TAG of the chylomicron. The chylomicron particle decreases in size and increases in density.
• Apo-C, including C-II, are returned to the HDL creating a chyomicron remnant.
• The chylomicron remnant is then endocytosed by a hepatocyte when Apo-E binds to specific lipoprotein receptors.
• Lysosomal hydrolyic enzymes degrade the chylomicron remnant components to cholesterol, amino acids, and fatty acids; the lipoprotein receptors are recycled.

Question 28

Summarize the VLDL/LDL life cycle (very low density lipoprotien/low density lipoprotein.)

Answer 28

• VLDLs are produced by the liver and secreted into the blood by
  as nascent particles containing Apo B-100.
• They obtain Apo E and Apo C-II from HDL particles. Some TAGS are transferred from VLDL to HDL in exchange for cholesterol esters in a reaction catalyzed by cholesterol ester transfer protein (CETP).
• Lipoprotein lipase (LPL) is activated by apo C-II. LPL hydrolyzes the TAG of the VLDL. The VLDL particle decreases in size and increases in density.

-VLDL is converted to LDL in the blood with Intermediate-density
lipoproteins (IDLs) or VLDL remnants observed during the transition.

• Apo-CII and Apo-E are returned to HDL particles.
• The LDL particle binds to a specific receptor on the surface of hepatocytes and extra-hepatic tissue, and is endocytosed.

Question 29

How does hepatic steatosis develop?

Answer 29

Nonalcoholic fatty liver occurs in conditions where there is an imbalance between TAG synthesis and secretion of VLDL.

Question 30

What are the isoforms of ApoE-2? What is the significance of each?

Answer 30

Apo E is present in 3 common isoforms. E3 is the most common and E2 the least; the other is E4. Apo E-2 binds poorly to receptors. Patients who are homozygous for E2 are deficient in clearance of chylomicrons or IDL. These people have familial Type III hyperlipoproteinemia (or familial dysbetalipoproteinemia) with hypercholesterolemia and premature atherosclerosis. The E-4 isoform confers increased susceptability and decreased age of onset for the lateonset form of Alzheimer’s Disease. The mechanism for this relationship remains unknown.

Question 31

Describe the uptake and degredation of LDL particles.

Answer 31

• LDL receptors are glycosylated transmembrane proteins which are clustered in clathrin coated pits. Apo B-100 and Apo E are recognized by these receptors.
• After binding the LDL-receptor complex is endocytosed with the assistance of clathrin in forming the coated vesicle. The coated vesicle losses its clathrin coat and fuses with other such vesicles to form endosomes.
• The pH of the endosome drops based on ATP dependent proton pumping into the endosome. This uncouples the receptor from the LDL particle. They separate into disitnct areas of what is called the Compartment for Uncoupling Receptor and Ligand (CURL).
• The receptors are recycled to the plasma membrane and the endosome fuses with a lysosome. Lysosomal hydrolases degrade the LDL (or chylomicron or IDL particles) releasing amino acids, fatty acids, cholesterol and phospholipids.

Question 32

What diseases are associated with the LDL receptor?

Answer 32

• A deficiency of LDL receptor causes elevated plasma LDL-cholesterol. Patients with this deficiency have Type II hyperlipidemia (Familial hypercholesterolemia or FH). There are many point mutations and deletions associated (~353.)
• Autosomal dominant hypercholesterolemia can be caused by increased activity of a protease that degrades the LDL receptor (proprotein convertase subtilisin/kexin type 9 or PCSK9.)

Question 33

Describe the formation of HDL particles, and their basic function .

Answer 33

• HDL particles are formed in blood by addition of lipids to apo A-1 which is synthesized in the liver and intestine and secreted into the blood.
• Nascent HDLs are discoid in shape and contain primarily phospholipids , and Apo A, C, and E. They take up cholesterol from peripheral tissues and return it to the liver as cholesterol esters
• When HDL takes up cholesterol it is esterified by lecithin:cholesterol acyl transferase (LCAT)
• HDL functions include serving as a circulating supplier of apo C-II and Apo E, and transporting cholesterol from peripheral tissues back to the liver.

Question 34

What are the steps involved in reverse cholesterol transport?

Answer 34

• The efflux of cholesterol from peripheral tissues is catalyzed by ABCA1 transporter also known as the cholesterol efflux regulatory protein (CERP.)
• in HDL LCAT is activated by Apo A-I and esterification of cholesterol maintains the cholesterol gradient allowing further uptake of cholesterol from peripheral tissues to HDL. As HDL picks up CE it converts from the discoidal nascent HDL to the relatively cholesterol poor HDL3 and then to the CE rich HDL2 particle that carries the CE to the liver. CETP exchanges CE from HDL to VLDL with concurrent exchange TAG from VLDL to HDL. This relieves product inhibition of LCAT. As VLDLs are catabolized to LDL the CEs transferred by CETP are taken up by the LDL receptor.
• The uptake of cholesterol esters from the liver involves the SR-B1 (scavenger receptor class B type 1) that binds HDL on the surface of hepatocytes and selectively takes up the cholesterol esters from the particle.

Question 35

What is Tangier disease?

Answer 35

Tangier disease (familial hypoalphalipoproteinemia) results from a rare inherited deficiency in ABCA1 (CREP) which leads to absence of HDL particles because of degradation of lipid-free apo A-I.

Question 36

Describe the reaction catalyzed by LCAT.

Answer 36

LCAT (lecithin–cholesterol acyltransferase) is activated by Apo A-I and transfers a fatty acid from carbon 2 of lecithin (phosphatidyl choline) to cholesterol producing a hydrophobic cholesterol ester (CE) and lyso-PC.

Question 37

How is cholesterol stored in cells?

Answer 37

If the cholesterol is not needed immediately for a synthetic or structural purpose it can be esterified by Acyl CoA :cholesterol acyl transferase (ACAT). The resulting cholesterol ester can be stored in the cell. The activity of ACAT is increased by the presence of an oversupply of intracellular cholesterol.

Question 38

Describe the funtion of CETP.

Answer 38

CETP catalyzes the exchange of TAG from VLDL with cholesterol ester from HDL.

Question 39

Explain the process of foam cell formation.

Answer 39

• Macrophages possess high levels of scavenger receptor activity. These scavenger receptors, known as scavenger receptor class A (SR-A), can bind a range of ligands causing endocytosis of modified LDL where the lipid or apo B have undergone oxidative damage.
• The scavenger receptor is not regulated by intracellular cholesterol concentration, thus the macrophages continue to consume excess oxidized LDL, and they become foam cells.
• The foam cells participate in plaque formation.

Question 40

Explain how thrombi develop.

Answer 40

-As a plaque within a blood vessel matures a cap forms over its
expanding “roof” partially occluding the vascular lumen.
- Vascular smooth muscle cells migrate from the tunica media to the subintimal space and secrete plaque matrix materials and
metalloproteases that thin the fibrous cap.
- The thinning continues until the capruptures exposing the cap contents to procoagulants within the circulation.

Question 41

LDL-R (location and function)

Answer 41

The LDL-R is expressed in most cells and regulates the entry of LDL into cells. It recognizes ApoB100 and ApoE. Tight control mechanisms alter its expression on the cell surface, depending on need.

Question 42

LDLR-Related Protein (Function)

Answer 42

Also known as the apoE receptor or the CMR receptor. Mediates the uptake of CM remnants and VLDL; preferentially recognize apoE but not apoB. Therefore, it mediates the metabolism of the major apoE containing LPs, including, CMR and IDL, but is not involved in LDL metabolism.

Question 43

Scavenger receptor B family (function and location)

Answer 43

Includes CD36 and SR-B1 . Uptake of cholesterol from HDL by the liver and steroid producing cells depends in part on the scavenger receptor class (SR-B1).